1. Field of the Invention
This invention concerns agonist anti-trkC monoclonal antibodies. It further concerns the use of the agonist antibodies in the prevention and/or treatment of cellular degeneration, including nerve cell damage associated with acute nervous cell system injury and chronic neurodegenerative diseases, including peripheral neuropathy.
2. Description of the Related Art
Neurotrophins are a family of small, basic proteins, which play a crucial role in the development and maintenance of the nervous system. The first identified and probably best understood member of this family is nerve growth factor (NGF), which has prominent effects on developing sensory and sympathetic neurons of the peripheral nervous system (Levi-Montalcini, R. and Angeletti, P. U., Physiol. Rev. 48, 534-569 [1968]; Thoenen, H. et al., Rev. Physiol. Biochem. Pharmacol. 109, 145-178 [1987]). Although NGF had been known for a long time, including a homolog from the mouse submandibular gland, the mature, active form of which is often referred to as - or 2.5S NGF, it was only many years later that sequentially related but distinct polypeptides with similar functions were identified.
The first in line was a factor called brain-derived neurotrophic factor (BDNF), which was cloned and sequenced by Leibrock, J. et al. (Nature 341, 149-152 [1989]). This factor was originally purified from pig brain (Barde, Y. A. et al., EMBO J. 1, 549-553 [1982]), but it was not until its cDNA was cloned and sequenced that its homology with NGF became apparent. The overall amino acid sequence identity between NGF and BNDF is about 50%. In view of this finding, Leibrock et al. speculated that there was no reason to think that BDNF and NGF should be the only members of a family of neurotrophins having in common structural and functional characteristics.
Indeed, further neurotrophins closely related to -NGF and BDNF have since been discovered. Several groups identified a neurotrophin originally called neuronal factor (NF), and now referred to as neurotrophin-3 (NT-3) (Ernfors et al., Proc. Natl. Acad. Sci. USA 87, 5454-5458 (1990); Höhn et al., Nature 344, 339 [1990]; Maisonpierre et al., Science 247, 1446 [1990]; Rosenthal et al., Neuron 4, 767 [1990]; Jones and Reichardt, Proc. Natl. Acad. Sci. USA 87, 8060-8064 (1990); Kaisho et al., FEBS Lett. 266, 187 [1990]. NT-3 shares about 50% of its amino acids with both -NGF and BDNF (NT-2). Neurotrophins-4 and -5 (NT-4 and NT-5), have been added to the family (U.S. Pat. No. 5,364,769 issued Nov. 15, 1994; Hallbook, F. et al., Neuron 6, 845-858 [1991]; Berkmeier, L. R. et al., Neuron 7, 857-866 [1991]; lp et al., Proc. Natl. Acad. Sci USA 89, 3060-3064 [1992]). The mammalian molecule initially described by Berkmeier et al supra, which was subsequently seen to be the homolog of Xenopus NT-4, is usually referred to as NT-4/5. In addition, there is an acidic homologous protein described in mammals which is referred to as NT-6 (Berkemeir, et al., Somat. Cell Mol. Genet 18(3):233-245 [1992]). More recently, another homologus protein from the fish, Xiphophorus has also been labeled NT-6 (Gotz et al., Nature 372:266-269 [1994]). There are two proteins described in the literature as NT-7, one cloned from the carp, Cyprinus, (Lai, et al., Mol. Cell. Neurosci. 11(1-2):64-76 [1998]) and one from the zebrafish, Danio (Nilsson et al., FEBS Letters 424(3):285-90 [1998]). None of these last three described fish neurotrophins has been described outside fish, and their relationship to any mammalian neurotrophins is unclear. The amino acid sequence of zebrafish neurotrophin-7 (zNT-7) is more closely related to that of fish nerve growth factor (NGF) and neurotrophin-6 (NT-6) than to that of any other neurotrophin. zNT-7 is, however, equally related to fish NGF and NT-6 (65% and 63% amino acid sequence identity, respectively) indicating that it represents a distinct neurotrophin sequence. zNT-7 contains a 15 amino acid residue in a beta-turn region in the middle of the mature protein. Recombinant zNT-7 was able to bind to the human p75 neurotrophin receptor and to induce tyrosine phosphorylation of the rat trkA receptor tyrosine kinase, albeit less efficiently than rat NGF. zNT-7 did not interact with rat trkB or trkC, indicating a similar receptor specificity as NGF. We propose that a diversification of the NGF subfamily in the neurotrophin evolutionary tree occurred during the evolution of teleost fishes which in the appearance of several additional members, such as zNT-7 and NT-6, is structurally and functionally related to NGF.
Neurotrophins, similarly to other polypeptide growth factors, affect their target cells through interactions with cell surface receptors. According to our current knowledge, two kinds of transmembrane glycoproteins serve as receptors for neurotrophins. Equilibrium binding studies have shown that neurotrophin-responsive neurons possess a common low molecular weight (65-80 kDa), low affinity receptor (LNGFR), also termed as p75NTR or p75, which binds NGF, BDNF, and NT-3 with a KD of 2×10−9 M, and large molecular weight (130-150 kDa), high affinity (KD in the 10−11 M) receptors, which are members of the trk family of the receptor tyrosine kinases.
The first member of the trk receptor family, trkA, was initially identified as the result of an oncogenic transformation caused by the translocation of tropomyosin sequences onto its catalytic domain (Martin-Zanca et al., Mol. Cell. Biol. 9(1):24-33 [1989]). Later work identified trkA as a signal transducing receptor for NGF. Subsequently, two other related receptors, mouse and rat trkB (Klein et al., EMBO J. 8, 3701-3709 [1989]; Middlemas et al., Mol. Cell. Biol. 11, 143-153 [1991]; EP 455,460 published 6 Nov. 1991) and porcine, mouse and rat trkC (Lamballe et al., Cell 66, 967-979 [1991]; EP 522,530 published 13 Jan. 1993), were identified as members of the trk receptor family. The structures of the trk receptors are quite similar, but alternate splicing increases the complexity of the family by giving rise to two known forms of trkA, three known forms of trkB (two without functional tyrosine kinase domains) and at least four forms of trkC (several without functional tyrosine kinase domain, and two with small inserts in the tyrosine kinase domain).
The role of the p75 and trk receptors is controversial. It is generally accepted that trk receptor tyrosine kinases play an important role in conferring binding specificity to a particular neurotrophin, however, cell lines expressing trkA bind not only NGF but also NT-3 and NT-415 (but not BDNF), trkB expressing cells bind BDNF, NT-3, NT-4, and NT-415 (but not NGF), in contrast to trkC-expressing cells which have been reported to bind NT-3 alone (but not the other neurotrophins). Furthermore, it has been shown in model systems that the various forms of trk receptors, arising from alternate splicing events, can activate different intracellular signalling pathways, and therefore presumably mediate different physiological functions in vivo. It is unclear whether cells expressing a given trk receptor in the absence of p75 bind neurotrophins with low or high affinity (Meakin and Shooter, Trends Neurosci. 15, 323-331 [1992]).
Published results of studies using various cell lines are confusing and suggest that p75 is either essential or dispensable for neurotrophin responsiveness. Cell lines that express p75 alone bind NGF, BDNF, NT-3, and NT-4 with similar low affinity at equilibrium, but the binding rate constants are remarkably different. As a result, although p75-binding is a common property of all neurotrophins, it has been suggested the p75 receptor may also play a role in ligand discrimination (Rodriguez-Tebar et al., EMBO J. 11, 917-922 [1992]). While the trk receptors have been traditionally thought of as the biologically significant neurotrophin receptors, it has recently been demonstrated that in melanoma cells devoid of trkA expression, NGF can still elicit profound changes in biological behavior presumably through p75 (Herrmann et al., Mol. Biol. Cell 4, 1205-1216 [1993]). Davies et al. (Neuron 11, 565-574 [1993]) reported the results of studies investigating the role of p75 in mediating the survival response of embryonic neurons to neurotrophins in a model of transgenic mice carrying a null mutation in the p75 gene. They found that p75 enhances the sensitivity of NGF-dependent cutaneous sensory neurons to NGF. There have now been many studies showing that p75 is capable of mediating at least some of the biological effects of the neurotrophins. The field is still somewhat controversial, but p75 signaling has been implicated in controlling cell death, and neurite outgrowth. (Barker, P A, Cell Death Diff. 5:346-356 [1998]; Bredesen et al., Cell Death Diff. 5:357-364 [1998]; Casaccia-Bonnefil, et al., Cell Death Diff. 5:357-364 [1998]; Raoul et al., Curr. Op. Neurobiol. 10:111-117 [2000]; Davies, A M, Curr. Biol. 10:R198-R200 [2000]). Importantly, stimulation of p75 has been shown to modify the effects of stimulating trkC (Hapner, et al., Developm. Biol. 201:90-100 [1998]).
The extracellular domains of full-length native trkA, trkB and trkC receptors have five functional domains, that have been defined with reference to homologous or otherwise similar structures identified in various other proteins. The domains have been designated starting at the N-terminus of the amino acid sequence of the mature trk receptors as 1) a first cysteine-rich domain extending from amino acid position 1 to about amino acid position 32 of human trkA, from amino acid position 1 to about amino acid position 36 of human trkB, and from amino acid position 1 to about amino acid position 48 of human trkC; 2) a leucine-rich domain stretching from about amino acid 33 to about amino acid to about amino acid 104 in trkA; from about amino acid 37 to about amino acid 108 in trkB, and from about amino acid 49 to about amino acid 120 in trkC; 3) a second cysteine-rich domain from about amino acid 105 to about amino acid 157 in trkA; from about amino acid 109 to about amino acid 164 in trkB; and from about amino acid 121 to about amino acid 177 in trkC; 4) a first immunoglobulin-like domain stretching from about amino acid 176 to about amino acid 234 in trkA; from about amino acid 183 to about amino acid 239 in trkB; and from about amino acid 196 to about amino acid 257 in trkC; and 5) a second immunoglobulin-like domain extending from about amino acid 264 to about amino acid 330 in trkA; from about amino acid 270 to about amino acid 334 in trkB; and from about amino acid 288 to about amino acid 351 in trkC.
Neurotrophins exhibit actions on distinct, but overlapping, sets of peripheral and central neurons. These effects range from playing a crucial role in ensuring the survival of developing neurons (NGF in sensory and sympathetic neurons) to relatively subtle effects on the morphology of neurons (NT-3 on purkinje cells). These activities have led to interest in using neurotrophins as treatments of certain neurodegenerative diseases. NT-3 has also been found to promote proliferation of peripheral blood leukocytes and, as a result, it has been suggested that NT-3 can be used in the treatment of neutropenia, infectious disease and tumors (U.S. Pat. No. 6,015,552 issued on Jun. 18, 2000).
The roles of neurotrophins in regulating cardiovascular development and modulating the vascular response to injury have also been investigated (Donovan et al., Nature Genetics 14:210-213 [1996]; Donovan et al., A.J. Path. 147:309-324 [1995]; Kraemer et al., Arteriol. Thromb. and Vasc. Biol. 19:1041-1050 [1999]). Neurotrophins have been described as potential therapeutics for regulating angiogenesis and vascular integrity (PCT Publication WO 00/24415, published May 4, 2000).
Despite their promise in the treatment of cellular degeneration, such as occurs due to neurodegenerative disease and acute neuronal injuries, and potentially angiogenesis, neurotrophins have several shortcomings. One significant shortcoming is the lack of specificity. Most neurotrophins cross-react with more than one receptor. For example NT-3, the preferred ligand of the trkC receptor tyrosine kinase, also binds to and activates trkA and trkB (Barbacid, J. Neurobiol. 25:1386-1403 [1994]; Barbarcid, Ann. New York Acad. Sci. 766:442-458 [1995]; Ryden and Ibanez, J. Biol. Chem. 271:5623-5627 [1996]; Belliveau et al., J. Cell. Biol. 136:375-388 [1997]; Farinas et al., Neuron 21:325-334 [1998]). As a result, it is difficult to devise therapies that target a specific population of neurons. Another limitation of neurotrophin therapy is that neurotrophins, including NT-3 are known to elicit hyperalgesia (Chaudhry, et al., Muscle and Nerve 23:189-192 [2000]). In addition, some neurotrophins such as NT-3 have poor pharmacokinetic and bioavailability properties in rodents, which raise serious questions about their human clinical applications (Haase et al., J. Neurol. Sci. 160:S97-S105 [1998], dosages used in Helgren et al., J. Neurosci. 17(1):372-82 [1997], and data below).
Accordingly, there is a great need for the development of new therapeutic agents for the treatment of neurodegenerative disorders and acute nerve cell injuries that are devoid of the known shortcomings of neurotrophins.